The invention relates generally to culturing pluripotent cells, and more particularly to chemically defined (i.e., synthetic) surfaces for long-term growth and maintenance (i.e., self-renewal) of pluripotent cells.
Pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells), have at least two characteristics that distinguish them from other types of cells. The first characteristic is that they are self-renewing, and thus are capable of growing indefinitely without differentiating. The second characteristic is that they can differentiate into cells of all three germ layers (i.e., endoderm, mesoderm, and ectoderm). See, e.g., Evans M & Kaufman M, “Establishment in culture of pluripotential cells from mouse embryos,” Nature 292:154-156 (1981), incorporated herein by reference as if set forth in its entirety.
One difficulty in working with pluripotent cells is developing standardized culture conditions for these cells without requiring the use of animal products or products such as serum, which tend to vary from batch to batch, to maintain the characteristics noted above. Important aspects of culturing these cells, therefore, are not only the medium in which they are grown, but also the surface upon which they are cultured.
Of particular interest herein are surfaces for culturing pluripotent cells, as these cells require adhesion/attachment to a surface to maintain the characteristics noted above. Although much information is available on chemically defining the constituents for culture medium for these cells, considerably less information is available on chemically defining the constituents of the surfaces and cell-substrate attachment for their survival and growth.
Initially, pluripotent cells were cultured on gelatin-coated surfaces containing mouse embryonic fibroblasts (MEFs) or other feeder cells. See, e.g., Amit M, et al., “Human feeder layers for human embryonic stem cells,” Biol. Reprod. 68:2150-2156 (2003); Lee J, et al., “Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition,” Biol. Reprod. 72:42-49 (2005); and Thomson J, et al., “Embryonic stem cell lines derived from human blastocysts,” Science 282:1145-1147 (1998). Pluripotent cells, however, do not grow on top of feeder cells, but instead tend to occupy the exposed gelatin-coated surface. As the cells proliferate, the growing colony pushes the MEFs away. See, e.g., Imreh M, et al., “Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system,” Stem Cells Dev. 13:337-343 (2004).
The art recognized that pluripotent cells can be cultured on a gelatin-coated surface in the presence of secreted factors from feeder cells, allowing the cells to be cultured in the absence of feeder cell layers (i.e., feeder-free). For example, feeder cell layers can be avoided through the use of “conditioned medium” (CM), which is medium in which feeder cells were cultured. However, culture of pluripotent cells on gelatin-coated surfaces in CM can lead to rapid differentiation of the cells. See, e.g., Xu C, et al., “Feeder-free growth of undifferentiated human embryonic stem cells,” Nat. Biotechnol. 19:971-974 (2001).
More recently, the art recognized that feeder cell layers also can be avoided by using a chemically defined culture medium (i.e., a complete medium), in which each constituent of the medium is fully disclosed and characterized. See, e.g., Ludwig T, et al., “Feeder-independent culture of human embryonic stem cells,” Nat. Methods 3:637-646 (2006); and Ludwig T, et al., “Derivation of human embryonic stem cells in defined conditions,” Nat. Biotechnol. 24:185-187 (2006), each of which is incorporated herein by reference as if set forth in its entirety.
One should not, however, overlook the role of surface attachment for successful pluripotent cell maintenance and growth. In this regard, feeder cell layers can be avoided through the use of a commercially produced extracellular matrix (ECM) material, such as Matrigel®. Matrigel®, however, contains indeterminate (i.e., undefined) quantities of murine extracellular matrix proteins, such as laminin, collagen and entactin. Additionally, there is batch to batch variation within Matrigel® and other unknown components such as, growth factors. Other ECM materials that can be used for pluripotent cell culture include vitronectin, fibronectin and laminin.
Chemically defined surfaces for pluripotent cells have been described (see, e.g., Derda R, et al., “Defined substrates for human embryonic stem cell growth identified from surface arrays,” ACS Chem. Biol. 2:347-355 (2007); and Gerecht S, et al., “Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells,” Proc. Natl. Acad. Sci. USA 104:11298-11303 (2007), but these surfaces have not yet proven effective for long-term growth and maintenance of pluripotent cells. Specifically, cells grown on these surfaces for several weeks form heterogeneous cell populations of undifferentiated and differentiated cells, which can be challenging to separate from one another. Bendall et al. “IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.” Nature (2007) 448:1015-1021 (2007). In addition, these surfaces typically rely on ECM proteins from animal or human sources. See, e.g., Amit M, et al., “Feeder layer- and serum-free culture of human embryonic stem cells,” Biol. Reprod. 70:837-845 (2004); Braam S, et al., “Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self renewal via αVβ5 integrin,” Stem Cells [Epub ahead of print, Jul. 17, 2008]; and Xu et al., supra.
As such, the art desires insoluble substrates with chemically defined surfaces and culture conditions for pluripotent cells that support their long-term growth and maintenance.
Further, there is a great need for methods for differentiating pluripotent cells on defined substrates and separating the differentiated cells from undifferentiated pluripotent cells.